Haemophilus somnus is a Gram negative bacterium which is related to several Actinobacillus species and appears to be identical to Histophilus ovis and Haemophilus agni (Philbey et al., Aust. Vet. J. (1991) 88:387-390). H. somnus causes a number of disease syndromes in animals. The bacterium is commonly associated with thromboembolic meningoencephalitis (ITEME), septicemia followed by sudden death, arthritis, and pneumonia (Corbeil, L. B., Can. J. Vet. Res. (1990) 54:S57-S62; Harris, F. W., and Janzen, E. D., Can. Vet. J. (1990) 30:816-822; Humphrey, J. D., and Stephens, L. R., Vet. Bull. (1983) 53:987-1004). These diseases can cause significant economic losses to the farm industry.
Current vaccines are either based on killed whole cells or on outer membrane protein (OMP) preparations. (See, e.g. U.S. Pat. Nos. 4,981,685, 4,877,613 and Stephens et al., Am. J. Vet. Res. (1984) 45:234-239). However, whole cell bacterins and surface protein extracts often contain immunosuppressive components which can render animals more susceptible to infection.
None of the above H. somnus preparations is enriched for iron-regulated proteins which are produced in iron-depleted environments. Certain iron-regulated proteins are immunogenic. For example, passive immunization with antibodies raised against iron-regulated outer membrane protein preparations has been shown to protect turkeys from Escherichia coli septicemia (Bolin, C. A., and Jensen, A. E., Infect. Immun. (1987) 55:1239-1242) and naturally occurring antibodies in human sera have been shown to react with iron-regulated outer membrane proteins of E. coli (Griffiths et al., Infect. Immun. (1985) 47:808-813). An iron-regulated protein has been identified in H. influenzae (Lee, B. C., Infect. Immun. (1992) 60:810-816).
In addition to Haemophilus infections, respiratory diseases associated with pathogenic microorganisms, particularly Pasteurella, and various stresses, such as transportation and overcrowding, are prevalent among feedlot cattle. One such disease is known as shipping fever which is characterized by sudden onset and pneumonia. Various bacteria and viruses have been isolated from affected animals including Pasteurella (particularly P. haemolytica and P. multicoda), bovine herpes virus 1, parainfluenza-3 virus, bovine respiratory syncytial virus and Mycoplasma species. For a general background on shipping fever see, Yates, W. D. G., Can. J. Comp. Med. (1982) 46:225-263.
P. haemolytica also causes enzootic pneumonia and can infect a wide range of animals, in addition to cattle, including sheep, swine, horses and fowl. P. haemolytica is also frequently found in the upper respiratory tract of healthy animals. Pneumonia develops when the bacteria infects the lungs of these animals. Protection against Pasteurella-associated diseases is therefore economically important to the agricultural industry.
There are two known biotypes of P. haemolytica designated A and T. There are also 12 recognized serotypes which have been isolated from ruminants. Biotype A, serotype 1 (referred to hereinafter as "A1") predominates in bovine pneumonia in North America (Shewen, P. E. and Wilkie, B. N., Am. J. Vet. Res. (1983) 44:715-719). However, antigens isolated from different serotypes appear to be somewhat cross-reactive (see, e.g., Donanchie et al., J. Gen. Micro. (1984) 130:1209-1216).
Previous vaccine preparations have included crude supernatant extracts from P. haemolytica (see, e.g., Shewen, P. E. and Wilkie, B. N., in Can. J. Vet. Res. (1988) 52:30-36). These culture supernatants, however, contain various soluble surface antigens of the bacterium and produce variable results when administered to animals. Other preparations include capsular extracts obtained via sodium salicylate extraction (see, e.g., Donanchie et al. J. Gen. Micro. (1984) 130:1209-1216; U.S. Pat. No. 4,346,074), saline extracted antigens (see, e.g., Lessley et al., Veterinary Immunology and Immunopathology (1985) 10:279-296; Himmel et al., Am. J. Vet. Res. (1982) 43:764-767), and modified live Pasteurella mutants.
Still other attempts at immunization have used a cytotoxin from P. haemolytica (see, e.g. Gentry et al., Vet. Immunology and Immunopathology (1985) 9:239-250; Conlon et al., Infect. Immun. (1991) 59:587-591; U.S. Pat. No. 4,957,739). This cytotoxin, which is a leukotoxin, is secreted by actively growing bacteria (Shewen, P. E., and Wilkie, B. N., Infect. Immun. (1987) 55:3233-3236). The gene encoding this cytotoxin has been cloned and expressed in bacterial cells (Lo et al., Infect. Immun. (1985) 50:667-67; U.S. Pat. Nos. 5,055,400; 4,957,739). Additionally, a truncated leukotoxin, which lacks the cytotoxic activity exhibited by the above cytotoxins, has been produced recombinantly and shown to be highly protective against shipping fever pneumonia (International Publication No. WO91/15237, published 17 Oct. 1991). However, none of these publications suggest the use of a leukotoxin in combination with an H. somnus antigenic preparation.